Methods and apparatus for optically imaging neuronal tissue and activity

ABSTRACT

The present invention provides a method and apparatus for distinguishing neuronal tissue from surrounding tissue, for distinguishing functional neuronal tissue from dysfunctional tissue, and for imaging of functional neuronal areas in cerebral cortex by detecting changes in the optical properties of the neuronal tissue following stimulation of neuronal activity.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation-in-part of U.S. patentapplication Ser. No. 08/073,353, filed Jun. 7, 1993 and issued as U.S.Pat. No. 5,465,718, which is a continuation-in-part of U.S. patentapplication Ser. No. 07/894,270, filed on Jun. 8, 1992 and issued asU.S. Pat. No. 5,438,989, which is a continuation-in-part of U.S. patentapplication Ser. No. 07/565,454 filed on Aug. 10, 1990 and issued asU.S. Pat. No. 5,215,095, all of which are incorporated herein byreference in their entirety.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to methods and apparatus for opticallyimaging neuronal tissue and the physiological events associated withneuronal activity. The methods and apparatus of the present inventionmay be used for optically imaging and mapping functional neuronalactivity, differentiating neuronal tissue from non-neuronal tissue,identifying and spatially locating dysfunctional neuronal tissue, andmonitoring neuronal tissue to assess viability, function and the like.

BACKGROUND OF THE INVENTION

Many experimental techniques have been applied to study the physiologyof the nervous system. Several of those techniques are described below.One of the applications for which the development of such techniques isessential is to assist a surgeon during surgery to avoid or reducedamage to functional neuronal tissue. Techniques currently used fordiagnostic and intraoperation assessment are also described below.

Hill and Keynes observed that the nerve from the walking leg of theshore crab (Carcinus maenas) normally has a whitish opacity caused bylight scattering, and that opacity changes evoked by electricalstimulation of that nerve were measurable. Hill, D. K. and Keynes, R.D., “Opacity Changes in Stimulated Nerve,” J. Physiol. 108:278-281(1949). Since the publication of those results, experiments designed tolearn more about the physiological mechanisms underlying the correlationbetween optical and electrical properties of neuronal tissue and todevelop improved techniques for detecting and recording activity-evokedoptical changes have been ongoing.

Several types of phenomenon relating to physiological neuronal activityhave been detected. Thermographic studies have detected thermalradiation changes that take place during neuronal activation usinginfrared imaging techniques. Spectrophotometric techniques have beenused to detect changes in absorption of the oxidizable fraction ofcytochrome oxidase in brain tissue. Spectroscopic techniques such aselectron microscopy and x-ray diffraction are not well-suited tostudying physiological activity in living neuronal tissue because of thehigh risk of tissue damage.

Many biomolecules fluoresce as a result of excitation with emr at thewavelength of the molecule's absorption band. This excitation causes themolecule to emit part of the absorbed energy at a different wavelength,and the emission can be detected using fluorometric techniques. Mostphysiological studies measuring intrinsic fluorescence have selected forNADH, which is an important intermediate in oxidative catabolism.Furthermore, NADH concentration in neuronal tissue is believed to becorrelated with neuronal activity. Upon excitation with ultravioletlight, NADH fluoresces at about 460 nm. Unfortunately, this techniquewould not be suitable for monitoring neuronal activity in humans,because illumination of in vivo neuronal tissue in vivo with ultravioletlight may cause serious tissue damage.

Another technique for detecting neuronal activity involvesadministration of a voltage-sensitive dye, whose optical propertieschange during changes in electrical activity of neuronal cells. Thespatial resolution achieved by this technique is near the single celllevel. For example, researchers have used the voltage-sensitive dyemerocyanine oxazolone to map cortical function in a monkey model.Blasdel, G. G. and Salama, G., “Voltage Sensitive Dyes Reveal a ModularOrganization Monkey Striate Cortex,” Nature 321:579-585, 1986. However,the use of these kinds of dyes would pose too great a risk for use inhumans in view of their toxicity. Furthermore, such dyes are bleached bylight and must be infused frequently.

Intrinsic changes in optical properties of cortical tissue have beenassessed by reflection measurements of tissue in response to electricalor metabolic activity. Grinvald, A., et al., “Functional Architecture ofCortex Revealed by Optical Imaging of Intrinsic Signals,” Nature324:361-364, 1986. Grinvald, et al., “Optical Imaging of NeuronalActivity, Physiological Reviews, Vol. 68, No. 4, October 1988. Grinvaldand his colleagues reported that some slow signals from hippocampalslices could be imaged using a CCD camera without signal averaging.

A CCD camera was used to detect intrinsic signals in a monkey model.Ts'o, D. Y., et al., “Functional Organization of Primate Visual CortexRevealed by High Resolution Optical Imaging,” Science 249:417-420, 1990.The technique employed by Ts'o et al. would not be practical for humanclinical use, since imaging of intrinsic signals was achieved byimplanting a stainless steel optical chamber in the skull of a monkeyand contacting the cortical tissue with an optical oil. Furthermore, inorder to achieve sufficient signal to noise ratios, Ts'o et al. had toaverage images over periods of time greater than 30 minutes per image.

Optically imaging neuronal and other types of tissue using techniquesand apparatus similar to those described herein is described in U.S.Pat. No. 5,215,095, allowed U.S. patent application Ser. No. 5,438,989,and allowed U.S. patent application Ser. No. 5,465,781, which areincorporated herein by reference in their entirety.

The mechanisms responsible for intrinsic signals are not wellunderstood. Possible sources of intrinsic signals include dilation ofsmall blood vessels, neuronal activity-dependent release of potassium,and swelling of neurons and/or glial cells caused, for example, by ionfluxes or osmotic activity. Light having a wavelength in the range of500 to 700 nm may also be reflected differently between active andquiescent tissue due to increased blood flow into regions of higherneuronal activity. Yet another factor which may contribute to intrinsicsignals is a change in the ratio of oxyhemoglobin and deoxyhemoglobin inblood.

One of the important applications for quantitative techniques thatidentify and assess neuronal tissue and function, both in the centraland the peripheral nervous system, is to provide information to medicalprofessionals prior to and during surgery. A neurosurgeon attempts tomap boundaries of dysfunctional tissue, so that dysfunctional tissue isremoved without affecting the surrounding tissue, and as much neuronalfunction as is possible is preserved. Neurological surgery is especiallyrisky, and precise resection of dysfunctional tissue without removingfunctional tissue is critical. It is also important for surgeons workingoutside the central nervous system to locate peripheral nerves and avoiddamaging them during other types of surgical procedures.

Current intraoperative techniques do not provide rapid or highresolution differentiation of dysfunctional neuronal tissue from normalneuronal tissue, or of neuronal tissue from surrounding tissue.Presently, electroencephalography (EEG) and electrocorticography (ECoG)techniques are used prior to and during neurosurgery for the purposes ofidentifying areas of abnormal neuronal activity. These measurementsprovide a direct measurement of the electrical activity in neuronaltissue.

One type of neurosurgical procedure which exemplifies these principlesis the surgical treatment of intractable epilepsy (that is, epilepsywhich cannot be controlled with medications). EEG and ECoG techniquesare typically used to identify epileptic foci. Intraoperative EEGtechniques involve placing an array of electrodes upon the surface ofthe cortex to detect electrical activity. This is done in an attempt tolocalize abnormal cortical activity of epileptic seizure discharge.

Although EEG techniques are of widespread use, hazards and limitationsare associated with these techniques. The size of the electrode surfaceand the distance between electrodes in an EEG array are large withrespect to the size of brain cells (e.g., neurons) with epileptic foci.Thus, current techniques provide poor spatial resolution (approximately1.0 cm) of the areas of abnormal cortical activity. Further, EEGtechniques do not provide a map of normal cortical function in responseto external stimuli (such as being able to identify a cortical areadedicated to speech function by recording electrical activity while thepatient speaks). A modification of this technique, called corticalevoked potentials, can provide some functional cortical mapping.However, the cortical evoked potential technique suffers from the samespatial resolution problems as the EEG technique.

The most common method of intraoperative localization of corticalfunction during neurosurgery is direct electrical stimulation of thecortical surface with a stimulating electrode. Using this technique, thesurgeon attempts to evoke either an observed motor response fromspecific parts of the body, or in the case of an awake patient, togenerate specific sensations or cause an interruption in the patient'sspeech output. Again, this technique suffers from the same problems asthe EEG technique because it offers only crude spatial localization offunction.

Possible consequences of the inaccuracies of all these techniques whenemployed, for example, to identify the portion of the cortex responsiblefor epileptic seizures, are either that a greater than necessary amountof cortical tissue is removed, possibly leaving the patient with adeficit in function, or that not enough tissue is removed, leaving thepatient uncured by the surgery. Despite these inadequacies, suchtechniques have been deemed acceptable treatment for intractableepilepsy.

A need in the art remains for methods and apparatus for opticallyimaging neuronal tissue which can precisely and quickly distinguishfunctional and dysfunctional (e.g., viable and nonviable) neuronaltissue, distinguish neuronal tissue from surrounding non-neuronaltissue, and map cortical neuronal function. Quantitative techniquesproviding the following capabilities would be desirable for assessingneuronal tissue: the ability to provide electrophysiological informationwith a high degree of spatial and temporal resolution; the ability tomonitor the activity of single neurons, as well as patterns of activityin larger areas of neuronal tissue, and the property of beingphysiologically non-invasive, i.e., providing data without requiringapplication of chemicals or penetration of mechanical devices, such asneuroelectrodes.

SUMMARY OF THE INVENTION

The methods and apparatus described herein can be used to identify areasof neuronal activity during surgical or diagnostic procedures, and tomonitor neuronal activity to assess tissue viability, function,recovery, degeneration and the like. For example, optical imagingtechniques of the present invention can be used by a surgeonintraoperatively to distinguish between functional and dysfunctionalneuronal tissue, or to distinguish between neuronal tissue andsurrounding non-neuronal tissue. In addition, the methods and apparatusof the present invention can be used to identify neuronal tissuededicated to important functions such as vision, movement, sensation,memory and language with a high degree of spatial resolution. Similarly,the methods and apparatus of the present invention can be used to detectareas of “abnormal” neuronal activity, whether that neuronal activity isunusually “high” or “low,” such as epileptic foci (“high”) or non-viableneuronal tissue (“low”). The present invention can also be used toidentify and locate individual nerves, for example, during neurosurgicalprocedures involving anastomoses of severed nerves or during other typesof surgery involving peripheral tissue, enabling the surgeon to avoiddamage to nerves. Although the optical imaging techniques disclosedherein are used principally for in vivo applications, they may be usedto monitor and assess neuronal activity for in vitro preparations aswell. The optical imaging techniques can be used to provide informationin “real time” and therefore can be employed intraoperatively.

The apparatus of the present invention employs an electromagneticradiation (emr) source for uniformly illuminating an area of interest,and an optical detector capable of detecting and acquiring data relatingto one or more optical properties of an area of interest. In a simpleform, the apparatus of the present invention may include an opticalfiber operably connected to an emr source that illuminates tissue, andanother optical fiber operably connected to an optical detector, such asa photodiode, that detects one or more optical properties of theilluminated tissue. The detector is used to obtain control datarepresenting the “normal” or “background” optical properties of an areaof interest, and then to obtain subsequent data representing the opticalproperties of an area of interest during neuronal activity, e.g.,stimulation of neuronal tissue, or during a monitoring interval. Thesubsequent data is compared to the control data to identify changes inoptical properties representative of neuronal activity. According to apreferred embodiment, the control, subsequent and comparison data arepresented in a visual format as images.

Various types of optical detectors may be used, depending on the opticalproperty being detected, the format of data being collected, certainproperties of the area of interest, and the type of application, e.g.,surgery, diagnosis, monitoring, or the like. In general, any type ofphoton detector may be utilized as an optical detector. The opticaldetector generally includes photon sensitive elements and opticalelements that enhance or process the detected optical signals. Numerousoptical detectors are known and may be used or adapted for use in themethods and apparatus of the present invention.

Changes in optical properties that may be indicative of neuronalactivity include, for example, reflection, refraction, diffraction,absorption, scattering, birefringence, refractive index, Kerr effect,and the like. The optical detection system may be incorporated in anapparatus for use external to the area of interest, or optical detectioncomponents may be mounted in an invasive or semi-invasive system, suchas an endoscope, laparoscope or the like.

High resolution optical imaging of physical changes indicative ofneuronal activity may be accomplished without using dyes or other typesof contrast enhancing agents according to the methods and apparatus ofthe present invention, as evidenced by the examples described herein.The optical imaging techniques of the present invention arephysiologically noninvasive, in that imaging of intrinsic signals doesnot require contacting the area of interest with any agents such asdyes, oils, devices, or the like. For particular applications, it may,however, be useful to administer contrast enhancing agents that amplifydifferences in an optical property being detected as a function ofneuronal activity prior to acquiring subsequent data and generating acomparison. The use of contrast enhancing agents is described in detail,with reference to optical imaging of tumor and non-tumor tissue, in U.S.Pat. Nos. 5,438,989 and 5,465,781 which are incorporated by referenceherein in their entirety. Suitable contrast enhancing agents includefluorescent and phosphorescent materials, dyes that bind to cellmembranes, optical probes that preferentially accumulate in blood or inthe intracellular space, phase resonance dye pairs, and the like.Detectors appropriate for use with such contrast enhancing agents arewell known in the art.

Numerous devices for acquiring, processing and displaying datarepresentative of one or more optical properties of an area of interestcan be employed. One preferred device is a video camera that acquirescontrol and subsequent images of an area of interest that can becompared to identify areas of neuronal activity or dysfunction.Examination of images provides precise spatial location of areas ofneuronal activity or dysfunction. Apparatus suitable for obtaining suchimages have been described in the patents incorporated herein byreference and are more fully described below. For most surgical anddiagnostic uses, the optical detector preferably provides images havinga high degree of spatial resolution at a magnification sufficient todetect single neuronal cells or nerve fiber bundles. Several images arepreferably acquired over a predetermined time period and combined, suchas by averaging, to provide control and subsequent images forcomparison.

Various data processing techniques may be advantageously used to assessthe data collected in accordance with the present invention. Comparisondata may be assessed or presented in a variety of formats. Processingmay include averaging or otherwise combining a plurality of data sets toproduce control, subsequent or comparison data sets. Images arepreferably converted from an analog to a digital form for processing,and back to an analog form for display.

Data processing may also include amplification of certain signals orportions of a data set (e.g., areas of an image) to enhance the contrastseen in data set comparisons, and to thereby identify areas of neuronalactivity and/or dysfunction with a high degree of spatial resolution.For example, according to one embodiment, images are processed using atransformation in which image pixel brightness values are remapped tocover a broader dynamic range of values. A “low” value may be selectedand mapped to zero, with all pixel brightness values at or below the lowvalue set to zero, and a “high” value may be selected and mapped to aselected value, with all pixel brightness values at or above the highvalue mapped to the high value. Pixels having an intermediate brightnessvalue, representing the dynamic changes in brightness indicative ofneuronal activity, may be mapped to linearly or logarithmicallyincreasing brightness values. This type of processing manipulation isfrequently referred to as a “histogram stretch” and can be usedaccording to the present invention to enhance the contrast of data sets,such as images, representing changes in neuronal activity.

Data processing techniques may also be used to manipulate data sets toprovide more accurate combined and comparison data. For example, patientmovement, respiration, heartbeat, seizure or reflex activity may shiftan area of interest during detection of optical properties and datacollection. It is important that corresponding data points in data sets(such as corresponding areas of an image) are precisely aligned toprovide accurate combined and comparison data. Such alignment may beaccomplished manually by a practitioner having specialized skill andexpertise, or using a variety of mathematical means. Optical markers maybe fixed at an area of interest and detected as the data is collected toaid in manual alignment or mathematical manipulation. Various processingtechniques are described below and in the patents incorporated herein byreference.

Inaccuracies and artifacts caused by patient movement during acquisitionof data can be reduced by mechanical means. According to a preferredembodiment, the emr source and the optical detector are provided as anintegral unit that is mountable to a patient during detection. Cranialposts are often provided when a patient has had substantial corticalinvolvement and may be used to mount an integrated emr source/detectorunit for detecting or mapping cortical neuronal activity or function.Likewise, an integrated unit including an emr source and an opticaldetector may be mounted in a relatively “fixed” condition in proximityto other areas of interest. Alternatively, and particularly for neuronalmonitoring applications, emr sources and/or optical detectors may beimplanted in the area(s) of interest and operably connected to externaldata processing devices.

Comparison data may be displayed in a variety of ways. Comparison datamay be displayed in a graphical format that highlights opticaldifferences indicative of neuronal activity. A preferred technique forpresenting and displaying comparison data is in the form of visualimages or photographic frames corresponding to the area of interest.This format provides a visualizable spatial location (two- orthree-dimensional) of neuronal activity and/or function that is usefulfor treatment, diagnosis and monitoring. To enhance and provide bettervisualization of contrast indicating neuronal activity or dysfunction,comparison images may be processed to provide an enhanced contrast greyscale or even a color image. A look up table (“LUT”) may be provided,for example, that converts the gray scale values for each pixel to adifferent (higher contrast) gray scale value, or to a color value. Colorvalues may map to a range of grey scale values, or color may be used todistinguish between positive-going and negative-going optical changes.In general, color-converted images provide higher contrast images thathighlight changes in optical properties representing neuronal activity,function or dysfunction.

In operation, an area of interest in a patient is illuminated withelectromagnetic radiation (emr) while one or a series of data points ordata sets representing one or more optical properties of the area ofinterest is acquired during an interval of “normal” neuronal activity.This data represents the control, or background data. A series of datasets is preferably combined, for example by averaging, to obtain acontrol data set. The control data set is stored for comparison withdata collected subsequently.

A subsequent data set representing the corresponding optical property isacquired during a subsequent time period. For monitoring applications,data may be collected at regular time intervals and monitored to detectaberrations from baseline values. For diagnostic or functional mappingapplications, a subsequent data set is collected during a period of(anticipated) neuronal activity or inhibition. Neuronal activity orinhibition may be induced by a “natural” occurrence such as a seizure orstroke, or it may be induced by administering a paradigm to the patientto stimulate an intrinsic neuronal signal. Intrinsic neuronal signalsmay be induced by direct electrical stimulation of neuronal tissue(whether in the central or peripheral nervous system), or by motoractivity, speech, thought, etc., that stimulates a specific corticalarea. During a monitoring interval or stimulation of an intrinsicsignal, one or a series of subsequent data sets, representing one ormore of the detected optical properties of the area of interest, isacquired. A series of subsequent data sets is preferably combined, forexample by averaging, to obtain a subsequent data set. The subsequentdata set is compared with the control data set to obtain a comparisondata set, preferably a difference data set. Comparison data sets canthen be examined for evidence of changes in optical propertiesrepresentative of neuronal activity or inhibition within the area ofinterest.

This technique can be used to identify areas of neuronal activity ordysfunction, and to accurately identify and map areas of specificneuronal function. Nerve dysfunction can be visualized and mapped as aportion of the nerve where an intrinsic signal from a stimulated nerveabruptly ends, or is altered, attenuated or diminished by comparing thecontrol data to the subsequent data. Likewise, viable neuronal tissuecan be distinguished from non-viable or nonfunctional neuronal tissue,and neuronal tissue can be distinguished from non-neuronal tissue. Byemploying the optical imaging techniques disclosed in this application,neuronal tissue can be monitored to detect changes in neuronal activitythat take place during development, neuronal trauma (e.g., seizure,stroke, and the like), recovery from neuronal trauma, administration oftherapeutic or diagnostic agents, and tissue transplantation andrecovery. Comparison images may be acquired and displayed in “real-time”for use during surgery, or over a more prolonged period, such as duringmonitoring of neuronal tissue viability, diagnostic or therapeuticagents, trauma, recovery, and the like. The optical imaging techniquesmay also be used to assess neuronal tissue viability and function invitro.

BRIEF DESCRIPTION OF THE DRAWINGS

This patent application contains at least one drawing executed in color.Copies of the patent with color drawings will be provided by the Patentand Trademark Office upon request and payment of the necessary fee.

The methods and apparatus of the present invention will be described ingreater detail with reference to the figures described below thatillustrate preferred embodiments.

FIG. 1A illustrates a view of human cortex just anterior to face-motorcortex with one recording (R) and two stimulating (S) electrodes, andfour sites (labeled 1, 2, 3, and 4) where average percent changes incorresponding optical properties were determined as described in Example1.

FIG. 1B shows plots of the percent optical change per second in thespatial regions of boxes 1 and 3 (as labeled in FIG. 1A). For bothregions, the peak change is during the fourth stimulation trial (at 8mA), in which the greatest amount of stimulating current had induced themost prolonged epileptiform afterdischarge activity. The changes withinbox 3 were greater and more prolonged than those of box 1. Box 3 wasoverlying the area of the epileptic focus.

FIG. 1C show plots of the percent optical change per second in thespatial regions of boxes 1 and 4 (as labeled in FIG. 1A). Box 1 overlaysand area of cortical tissue between the two stimulating electrodes, andbox 4 overlays a blood vessel. The optical changes within box 4 are muchlarger and in the opposite direction of box 1. Also, these changes aregraded with the magnitude of stimulating current and afterdischargeactivity. The changes in box 4 are most likely due to changes of theblood-flow rate within a blood vessel. This data demonstrates that themethods and apparatus of the present invention can be used tosimultaneously monitor cortical activity and blood-flow.

FIG. 1D shows plots of the percent optical change absorption per secondin the spatial regions of boxes 1 and 2 (as labeled in FIG. 1A). Notethat although these two areas are nearby each other, their opticalchanges are in the opposite direction during the first three stimulationtrials using 6 mA current. The negative going changes within the regionof box 2 indicate that the methods and apparatus of the presentinvention may be used to monitor inhibition of cortical activity as wellas excitation.

FIG. 2 illustrates spatial images of stimulation-induced epileptiformactivity. The images show comparisons between different degrees ofactivation illustrating both the spatial extent and amplitude of opticalchanges indicative of the extent of cortical activity. Specifically,FIG. 2 shows percentage difference images representative of varioustimes during two of the stimulation trials described in Example 1. Thetop 3 images (2A2, 2B2, and 2C2) are from stimulation trial 2, where 6mA cortical stimulation evoked a brief period of afterdischarge. Theseare compared to the bottom three images (2A4, 2B4, and 2C4), which arefrom stimulation trial 4, showing the optical changes evoked by corticalstimulation at 8 mA. FIGS. 2A2 and 2A4 compare control images duringrest. FIGS. 2B2 and 2B4 compare the peak optical changes occurringduring the epileptiform afterdischarge activity. FIGS. 2C2 and 2C4compare the degree of recovery 20 seconds after the peak optical changeswere observed. The magnitude of optical change is indicated by thegray-scale changes. Each image maps an area of cortex approximately 4 cmby 4 cm.

FIG. 3 shows eight percentage difference images from stimulation trial 2described in the previous two Figures. Each image is integrated over atwo second interval. The focal area of greatest optical change is in thecenter of images 3C, 3D, and 3E, indicating the region of greatestcortical activity. This region is the epileptic focus. The magnitude ofoptical change is indicated by the gray-scale bar on the right side ofthe Figure. The arrow beside this gray-scale indicates the direction ofincreasing amplitude. Each image maps an area of cortex approximately 4cm by 4 cm.

FIG. 4 illustrates a real-time sequence of dynamic changes ofstimulation-evoked optical changes in human cortex. FIG. 4, panels 4Athrough 4H, show eight consecutive percentage difference images. Eachimage is an average of 8 frames (<¼ second per image). The magnitude ofoptical change is indicated by the gray-scale changes. Each image mapsto an area of cortex that is approximately 4 cm by 4 cm. This FIG.demonstrates that the methods and apparatus of the present invention canbe used to map, in real time, dynamics of optical changes, and displaysuch information to a surgeon in an informative format.

FIG. 5 illustrates activation of somatosensory cortex by stimulation ofa peripheral nerve in an anesthetized rat (inducing afferent sensoryinput by directly stimulating the sciatic nerve in the hind limb of arat). The leftmost image,

FIG. 5A, is a gray-scale image of hind limb somatosensory cortex in ananesthetized rat. The magnification is sufficiently high so thatindividual capillaries can be distinguished (the smallest vesselsvisible in this image). The center image, FIG. 5B, is an image of apercentage difference control optical image during rest. The magnitudeof optical change is indicated by the gray-scale bar on the right sideof this image. The rightmost image, FIG. 5C, is a percentage differencemap of the optical changes in the hind limb somatosensory cortex duringstimulation of the sciatic nerve.

FIG. 6 illustrates functional mapping of human language (Broca's area)and tongue and palate sensory areas in an awake human patient asdescribed in Example 2. Images 6A1 and 6B1 are gray-scale images of anarea of human cortex, with left being anterior, right-posterior,top-superior, and the Sylvan fissure on the bottom. The two asterisks on6A1, 6B1, 6A2, and 6B2 serve as reference points for these images. Thescale bars in the lower right corner of 6A1 and 6B1 are equal to 1 cm.In 6A1, the numbered boxes represent sites where cortical stimulationwith electrical stimulating electrodes evoked palate tingling (1),tongue tingling (2), speech arrest-Broca's areas (3,4) and no response(11, 12, 17, 5, 6-7 premotor). Image 6A2 is a percentage differencecontrol image of the cortex during rest in one of the tongue wigglingtrials. The gray-scale bar on the right of 6A2 shows the relativemagnitude of the gray values associated with images 6A2, 6A3, 6B2 and6B3. Image 6A3 is a percentage difference map of the peak opticalchanges occurring during one of the tongue wiggling trials. Areasidentified as tongue and palate sensory areas by cortical stimulationshowed a large positive change. Suppression of baseline noise insurrounding areas indicated that, during the tongue wiggling trials,language-motor areas showed a negative-going optical signal. Image 6B2is percentage difference control image of the cortex during one of thelanguage naming trials. Image 6B3 is a percentage difference image ofthe peak optical change in the cortex during the language naming task.Large positive-going signals are present in Broca's area. Negative-goingsignals are present in tongue and palate sensory areas.

FIG. 7 shows time course and magnitude plots of dynamic optical changesin human cortex evoked in tongue and palate sensory areas and in Broca'sarea (language). This Figure shows the plots of the percentage change inthe optical absorption of the tissue within the boxed regions shown inFIG. 6, images 6A1 and 6B1, during each of the three tongue wigglingtrials and one of the language naming trials (see description of FIG.6).

FIG. 7A shows the plots during the three tongue wiggling trials averagedspatially within the boxes 1, 2, 3, and 4 as identified in FIG. 6A1.FIG. 7B shows the plots during one of the language naming trialsaveraged spatially within the boxes 1-7 and 17.

FIG. 8 illustrates an optical map of a cortical area important forlanguage comprehension (Wernicke's area) in an awake human.

FIG. 8A shows the cortical surface of a patient where the anatomicalorientation is left-anterior, bottom-inferior, with the Sylvan fissurerunning along the top. After optical imaging, all cortical tissue to theleft of the thick line was surgically removed. Sites #1 and #2 wereidentified as essential for speech (e.g., cortical stimulation blockedability of subject to name objects). At site #3, one naming error in 3stimulation trials was found. As the surgical removal reached the arealabeled by the asterisks on the thick line, the patient's languagedeteriorated. All the unlabeled sites in FIG. 8A had no errors whilenaming slides during cortical stimulation.

FIG. 8B shows an overlay of a percentage difference image over thegray-scale image of the cortex acquired during a language naming trial(see FIG. 6 for description of the language naming trial). The magnitudeof the optical change is shown by the gray-scale bar on the lower rightof the image.

FIG. 9 illustrates a time course and magnitude of dynamic opticalchanges in human cortex evoked in Wernicke's area (languagecomprehension).

FIG. 9A shows plots of percentage change in optical absorption of tissuewithin the boxed regions shown in FIG. 8. The plots of boxes 1 and 2overlay essential language sites, and boxes labeled 4, 5, and 6 overlaysecondary language sites. Each of these five sights showed significantchanges occurring while the patient was engaged in a language namingtask.

FIG. 9B shows percentage changes from the six unlabeled boxes shown inFIG. 8. There were no significant increases or decreases within theseanterior sites.

FIG. 10A is a gray-scale image of the cranial surface of a rat. Thesagittal suture runs down the center of the image. Box 1 lays over thesuspected region of brain tumor, and box 2 lays over normal tissue.

FIG. 10B is a difference image 1 second after indocyanine green dye hadbeen intravenously injected into the animal. The region containing tumortissue became immediately visible through the intact cranium.

FIG. 10C shows that 5 seconds after dye injection the dye can be seen toprofuse through both normal and tumor tissue.

FIG. 10D shows that 1 minute after dye injection, the normal tissue hadcleared the dye, but dye was still retained in the tumor region. Theconcentration of dye in the center of this difference image was dyecirculating in the sagittal sinus. Neuronal activity may likewise beimaged through other intact tissues, such as bone, duva, muscle,connective tissue, and the like.

FIG. 11 illustrates a grey-scale image of human cortex just anterior toface-motor cortex with one recording (R) and two stimulating (s)electrodes for applying stimulating current to induce epileptiform afterdischarge activity. Surface electrical signals were obtained byconventional EEG techniques. The cortex as illuminated with emr ofwavelengths greater than about 690 nm and the images presented in FIGS.6B-6E were acquired using a CCD camera as described herein. The imageswere processed to map regions of increasing (positive-going), decreasing(negative-going), and non-changing levels of cortical activity to thecolors red, blue and black, respectively. Each image maps to an area ofcortex that is approximately 4 cm by 4 cm.

FIG. 11A is a grey-scale image of a human cortex just anterior toface-motor cortex with two stimulating electrodes (s).

FIG. 11B is spatial map of baseline cortical activity prior toapplication of stimulating current for inducing epileptiformafterdischarge activity.

FIG. 11C is a spatial map of cortical activity during stimulation atstimulating electrodes (s) and the resulting epileptiform afterdischargeactivity.

FIG. 11D is a spatial map of cortical activity during an apparentquiescent period following the epileptiform afterdischarge activityinduced by stimulation at stimulating electrodes (s).

FIG. 11E is a spatial map of cortical activity of a period following thequiescent period represented by FIG. 11D.

FIGS. 11B-11E each correspond to an average of approximately 60 framesacquired at 30 Hz over a period of about 2 seconds.

FIG. 12 is a trace of an EEG recording of surface electrical signalsreceived by recording electrode (r) shown in FIG. 11A and correspondingto the baseline cortical activity of FIG. 11B (period A), the corticalactivity during stimulation and the resulting epileptiformafterdischarge activity of FIG. 11C (period B), the quiescent corticalactivity following the epileptiform afterdischarge activity of FIG. 11D(period C), and the subsequent cortical activity of FIG. 11E (period D).

FIG. 13 shows functional mapping of human language (Broca's area) andtongue and palate sensory areas in an awake human patient.

FIGS. 13A1 and 13B1 are gray-scale images of an area of human cortexwith left being anterior, right-posterior, top-superior, and the Sylvanfissure on the bottom. The numeral 34 in FIG. 13A1 (partly obscured)serves as reference point to FIG. 13B1 in which the numeral is mostlyobscured at the upper right edge of the Figure. Each image maps to anarea of cortex that is approximately 4 cm by 4 cm.

FIGS. 13A2 and 13B2 are spatial maps of cortical activity in the areasof human cortex shown in FIGS. 12A1 and 12B1 during, respectively, alanguage naming exercise and a tongue wiggling exercise. The images wereprocessed to map increasing, decreasing and constant levels of corticalactivity in the colors red, blue and black, respectively.

FIG. 14 is a simplified schematic diagram illustrating an apparatus ofthe present invention.

DETAILED DESCRIPTION OF THE INVENTION

Applicants' optical imaging methods and apparatus are described ingreater detail below with reference to certain preferred embodiments.Certain aspects of the optical imaging technique have been described ineven greater detail in the patents incorporated herein by reference. Thedetailed descriptions of certain preferred embodiments described hereinare not intended to limit the scope of applicants' invention as setforth in the appended claims.

Definitions

The following terms, as used in this specification and the appendedclaims, have the meanings indicated:

Area of Interest is an area of tissue that comprises the subject ofacquired data sets. The area of interest may, for example, be exposedtissue, tissue that underlies or is adjacent exposed tissue, or tissuecultured in vitro.

Arithmetic Loqic Unit (ALU) is a component that is capable of performinga variety of processing (e.g., mathematical and logic) operations (e.g.,sum, difference, comparison, exclusive or multiply by a constant, etc.)on a data set.

Control Data is data representing one or more optical properties of thearea of interest during a “normal” period or a predetermined period,such as prior to stimulation of an intrinsic signal. The control dataset establishes a “background” level of neuronal activity for comparisonwith a subsequently acquired data set.

Charge Coupled Device (CCD) is a type of optical detector that utilizesa photo-sensitive silicon chip in place of a pickup tube in a videocamera.

Comparison Data is data acquired by comparing subsequent data or dataacquired at a particular time, with control data, such as by adding,subtracting, or the like. The comparison data set is used to identifyand/or locate areas of neuronal activity.

Electromagnetic Radiation (emr) means energy having a wavelength of fromabout 450 to about 2500 nm. Emr illumination suitable for use in theoptical imaging methods described herein is in the visible and infraredregions.

Frame is a digitized array of pixels.

Frame Buffer is a component that provides storage of a frame, such as acontrol image, a subsequent image or a comparison image.

Geometric Transformations can be used to modify spatial relationshipsbetween data points in a data set, such as pixels in an image. Geometrictransformations are often called “rubber sheet transformations” becausethey can be viewed as the process of “printing” data, such as an image,on a sheet of rubber and stretching the sheet according to a predefinedset of rules. As applied to video imaging, subsequent images can beviewed as having been distorted due to movement and it is desirable to“warp” these images so that they are spatially aligned with the controlimages. Geometric transformations are distinguished from “pointtransformations” in that point transformations modify a pixel's value inan image based solely upon that pixel's value and/or location, and noother pixel values are involved in the transformation. Geometrictransformations are described in the publication Digital ImageProcessing, Gonzalez and Wintz, Addison-Wesley Publishing Co., Reading,1987.

Image is a frame or composition of frames representing one or moreoptical properties of an area of interest.

Intrinsic Signal is a detectable change in one or more opticalproperties of neuronal tissue caused by physiologic activity. Intrinsicsignals may be the related to membrane depolarization, glial cellswelling, ion flux across neuronal membranes, blood volume changes,blood oxygenation and deoxygenation tissue oxygenation and combinationsthereof.

Optical properties relate to various properties detectable in the usefulrange of emr (450-2500 nm) including but not limited to scattering(Rayleigh scattering, reflection/retraction, diffraction, absorption andextinction), birefringence, refractive index, Kerr effect and the like.

Optical Detector is a device capable of detecting one or more desiredoptical properties of an area of interest. Suitable optical detectorsinclude any type of photon detector, such as photodiodes,photomultiplier tubes, cameras, video cameras, CCD cameras, and thelike.

Optical Imaging refers to the acquisition, comparison, processing anddisplay of data representative of one or more optical properties of anarea of interest that indicate neuronal activity. Optical imaging mayinvolve acquisition processing and display of data in the form ofimages, but need not.

Paradigms cause a change in electrical activity of an area of neuronaltissue dedicated to a specific function, thus causing a change inneuronal activity reflected in the intrinsic signal. Ojemann, forexample, has described paradigms which produce neuronal activity inareas of the cortex dedicated to such functions as speech, language,vision, etc. Ojemann, “Functional Mapping of Cortical Language Areas inAdults—Intra—operative Approaches,” Electrical and Magnetic Stimulationof the Brain and Spinal Cord, edited by O. Devinsky, et al., RavenPress, Ltd., N.Y. N.Y. 1993. Administering a paradigm may also involvedirect electrical stimulation of neuronal tissue.

Pixels are the individual units of an image in each frame of a digitizedsignal. The intensity of each pixel is linearly proportional to theintensity of illumination before signal manipulation and corresponds tothe amount of emr (photons) being scattered from a particular area oftissue corresponding to that particular pixel. An image pixel is thesmallest unit of a digital image and its output intensity can be anyvalue. A CCD pixel is the smallest detecting element on a CCD chip andits analog output is linearly proportional to the number of photons itdetects.

Subsequent Data is data representing one or more optical properties ofan area of interest during a monitoring period or during or subsequentto stimulating an intrinsic signal.

Tissue means any cellular or multicellular mammalian component, whetheror not it has a distinct structure or function. Neuronal tissue, forexample, refers to individual neurons, bundles or collections ofneurons, as well as highly organized cortex.

Apparatus

The inventive methods employ an apparatus comprising a source of highintensity emr, an optical detector for acquiring data representative ofone or more optical properties of the area of interest, such as videosignals, and image processing capability. The apparatus may beconstructed as an integrated unit, or it may be used as a collection ofcomponents. The apparatus will be briefly described with reference tothe schematic diagram, illustrated in FIG. 14, and various componentsand features will then be described in greater detail.

FIG. 14 illustrates a human patient 10 whose neuronal tissue representsarea of interest 12. As is described in greater detail below, area ofinterest 12 may be fully or partially exposed, or imaging may beconducted through bone and/or dura with proper selection of emrwavelengths. During optical imaging, area of interest 12 is uniformlyilluminated by emr source 14 powered by regulated power supply 16. Emris preferably directed through an optical filter 18 prior to contactingarea of interest 12.

During optical imaging, a light gathering optical element 20, such as acamera lens, endoscope, optical fibers and photon detector 22 or thelike are placed to detect optical properties of area of interest 12.Signals representative of optical properties are processed, if desired,in a gain, offset component 24 and then conveyed to analog-to-digital(A/D) and digital signal processing hardware 26. Data representingoptical properties and particularly changes in optical properties, aredisplayed on display device 28. The optical detection, display andprocessing components are controlled by host computer 30.

An emr source is used for illuminating the area of interest duringacquisition of data representing one or more optical properties. Inaccordance with preferred methods of the present invention, the area ofinterest is typically cortical neuronal tissue or peripheral neuronaltissue. The emr source may be utilized to illuminate an area of interestdirectly, as when neuronal tissue is exposed during or in connectionwith surgery, or it may be utilized to illuminate an area of interestindirectly through adjacent or overlying tissue such as bone, dura,skin, muscle and the like.

The emr source employed in the present invention is preferably a highintensity, broad spectrum emr source, such as a tungsten-halogen lamp,laser, light emitting diode, or the like. Cutoff filters to selectivelypass all wavelengths above or below a selected wavelength may beemployed. A preferred cutoff filter excludes all wavelengths below about695 nm. Preferred emr wavelengths for imaging intrinsic signals include,for example, wavelengths of from about 450 nm to about 2500 nm, and mostpreferably, wavelengths of the near infrared spectrum of from about 700nm to about 2500 nm. Generally, longer wavelengths (e.g., approximately800 nm) are employed to measure deeper cortical activity. Selectedwavelengths of emr may also be used, for example, when various types ofcontrast enhancing agents are administered. The emr source may bedirected to the area of interest by a fiber optic means. One preferredarrangement provides emr through strands of fiber optic using a beamsplitter controlled by a D.C. regulated power supply (Lambda, Inc.).

The optical imaging methods of the present invention may also usefullyemploy non-continuous illumination and detection techniques. Forexample, short pulse (time domain), pulsed time, and amplitude modulated(frequency domain) illumination sources may be used in conjunction withsuitable detectors (See, Yodh, A., and Chance, B. Physics Today, March,1995). Frequency domain illumination sources typically comprise an arrayof multiple source elements, such as laser diodes, with each elementmodulated at 180° out of phase with respect to adjacent elements (see,Chance, B. et al., (1993) Proc. Natl. Acad. Sci. USA, 90, 3423-3427).Two-dimensional arrays, comprising four or more elements in twoorthogonal planes, can be employed to obtain two-dimensionallocalization information. Such techniques are described in U.S. Pat.Nos. 4,972,331 and 5,187,672 which are hereby incorporated by reference.

Time-of-flight and absorbance techniques (Benaron, D. A. and Stevenson,D. K. (1993) Science, 259, 1463-1466) may also be usefully employed inthe present invention. In yet another embodiment of the presentinvention, a scanning laser beam may be used in conjunction with asuitable detector, such as a photomultiplier tube, to obtain highresolution images of an area of interest.

Illumination with a part of the infrared spectrum allows for imagingintrinsic signals through tissue overlying or adjacent the area ofinterest, such as dura and skull. One exemplary infrared emr sourcesuitable for imaging through tissue overlying or adjacent the area ofinterest is a Tunable IR Diode Laser from Laser Photonics, Orlando, Fla.When using this range of far infrared wavelengths, the optical detectoris preferably provided as an infrared (IR) detector. IR detectors aremade from materials such as indium arsenide, germanium and mercurycadmium telluride and are generally cryogenically cooled to enhancetheir sensitivity to small changes in infrared radiation. One example ofan IR imaging system which may be usefully employed in the presentinvention is an IRC-64 infrared camera (Cincinnati Electronics, Mason,Ohio).

The area of interest must be evenly illuminated to effectively adjustthe signal over a full dynamic range, as described below. Nonuniformityof illumination is generally caused by fluctuations of the illuminationsource and intensity variations resulting from the three-dimensionalnature of the tissue surface. More uniform illumination can be providedover the area of interest, for example, by using diffuse lighting,mounting a wavelength cutoff filter in front of the optimal detectorand/or emr source, or combinations thereof. Fluctuation of theillumination source itself is preferably addressed by using a lightfeedback mechanism to regulate the power supply of the illuminationsource. In addition, a sterile, optically transparent plate may contactand cover the area of interest to provide a flatter, more even contour.The plate also diminishes tissue movement. Fluctuations in illuminationcan be compensated for by using image processing algorithms, includingplacing a constant shade gray image marker point at the area of interestas a control point.

The apparatus also comprises an optical detector for acquiring a signalrepresentative of one or more optical properties of the area ofinterest. Any photon detector may be employed as an optical detector.Specialized detectors suited for detecting selected optical propertiesmay be employed. One preferred optical detector for acquiring data inthe format of an analog video signal is a charge coupled device (CCD)video camera which produces an output video signal at 30 Hz having, forexample, 512 horizontal lines per frame using standard RS 170convention. One suitable device is a CCD-72 Solid State Camera (Dage-MTIInc., Michigan City, Ind.). Another suitable device is a COHU 6510 CCDMonochrome Camera with a COHU 6500 electronic control box (COHUElectronics, San Diego, Calif.). In some cameras, the analog signal isdigitized 8-bits deep on an ADI board (analog-to-digital board). The CCDmay be cooled, if necessary, to reduce thermal noise.

The optical imaging methods of the present invention may also usefullyemploy non-continuous illumination and detection techniques. Forexample, short pulse (time domain), pulsed time, and amplitude modulated(frequency domain) illumination sources may be used in conjunction withsuitable detectors (See, Yodh, A., and Chance, B. Physics Today, March,1995). Frequency domain illumination sources typically comprise an arrayof multiple source elements, such as laser diodes, with each elementmodulated at 180° out of phase with respect to adjacent elements (see,Chance, B. et al., (1993) Proc. Natl. Acad. Sci. USA, 90, 3423-3427).Two-dimensional arrays, comprising four or more elements in twoorthogonal planes, can be employed to obtain two-dimensionallocalization information. Such techniques are described in U.S. Pat.Nos. 4,972,331 and 5,187,672 which are hereby incorporated by reference.

Time-of-flight and absorbance techniques (Benaron, D. A. and Stevenson,D. K. (1993) Science, 259, 1463-1466) may also be usefully employed inthe present invention. In yet another embodiment of the presentinvention, a scanning laser beam may be used in conjunction with asuitable detector, such as a photomultiplier tube, to obtain highresolution images of an area of interest.

Image (data) processing is an important feature of the optical imagingtechniques and apparatus of the present invention. In use, for example,CCD apparatus is preferably adjusted (at the level of the analog signaland before digitizing) to amplify the signal and spread the signalacross the full possible dynamic range, thereby maximizing thesensitivity of the apparatus. Specific methods for detecting opticalsignals with sensitivity across a full dynamic range are described indetail in the patents incorporated herein by reference. Means forperforming a histogram stretch of the difference frames (e.g.,Histogram/Feature Extractor HF 151-1-V module, Imaging Technology,Woburn, Mass.) may be provided, for example, to enhance each differenceimage across its dynamic range. Exemplary linear histogram stretches aredescribed in Green, Digital Image Processing: A Systems Approach, VanNostrand Reinhold, N.Y., 1983. A histogram stretch takes the brightestpixel, or one with the highest value in the comparison image, andassigns it the maximum value. The lowest pixel value is assigned theminimum value, and every other value in between is assigned a linearvalue (for a linear histogram stretch) or a logarithmic value (for a loghistogram stretch) between the maximum and minimum values. This allowsthe comparison image to take advantage of the full dynamic range andprovide a high contrast image that clearly identifies areas of neuronalactivity or inactivity.

Noise (such as 60 Hz noise from A.C. power lines) is filtered out in thecontrol box by an analog filter. Additional adjustments may furtherenhance, amplify and condition the analog signal from a CCD detector.One means for adjusting the input analog signal is to digitize thissignal at video speed (30 Hz), and view the area of interest as adigitized image that is subsequently converted back to analog format.

It is important that data, such as consecutive images of a particulararea of interest, be aligned so that data corresponding to the samespatial location can be compared. If an averaged control image and asubsequent image are misaligned prior to comparison, artifacts will bepresent and the resulting comparison image will be more like a gradientimage that amplifies noise and edge information. Image misalignment canbe caused by patient motion, heartbeat and respiration. Large patientmovements may require a new orientation of the camera and acquisition ofa new averaged control image. It is possible, however, to compensate forsmall tissue movements by either mechanical or computational means, or acombination of both.

One way to relative movement of the optical detector and the area ofinterest is to rigidly secure the optical detector, and possibly the emrsource, to the skeletal frame of the patient, such as by posts mountedon the cranium. The optical detector and emr source may also be providedas an integral limit to reduce relative motion. Other means formonitoring the optical detector and the illumination source in aconstant orientation with respect to the area of interest may also beemployed.

Real-time motion compensation and geometric transformations may also beused to align corresponding data. Simple mechanical translation of dataor more complex (and generally more accurate) geometric transformationtechniques can be implemented, depending upon the input data collectionrate and amount and type of data processing. For many types of images,it is possible to compensate by a geometrical compensation whichtransforms the image by translation in the x-y plane. In order for analgorithm such as this to be feasible, it must be computationallyefficient (preferably implementable in integer arithmetic), memoryefficient, and robust with respect to changes in ambient light.

For example, functional control points can be placed in the area ofinterest and triangulation-type algorithms used to compensate formovements of these control points. Control points can be placed directlyin the area of interest, such as directly on the cortical surface.Goshtasby (“Piecewise Linear Mapping Functions for Image Registration”in Pattern Recognition vol. 19 pp 459-66, 1986) describes a methodwhereby an image is divided into triangular regions using controlpoints. A separate geometrical transformation is applied to eachtriangular region to spatially register each control point to acorresponding triangular region in a control image.

“Image warping” techniques may be employed whereby each subsequent imageis registered geometrically to the averaged control image to compensatefor movement. Image warping techniques described in, for example,Wolberg, “Digital Image Warping” IEEE Computer Society Press, LosAlimitos, Calif. 1990, may be used. Image warping techniques can furtherindicate when movement has become too great for effective compensationand a new averaged control image must be acquired.

The data processing function is generally operated and controlled by ahost computer. The host computer may comprise any general computer (suchas an IBM PC type with an Intel 386, 486 Pentium or similarmicroprocessor or Sun SPARC) that is interfaced with the emr sourceand/or optical detector directs data flow, computations, imageacquisition and the like. Thus, the host computer controls acquisitionand processing of data and provides a user interface.

According to a preferred embodiment, the host computer comprises asingle-board embedded computer with a VME64 interface, or a standard(IEEE 1014-1987) VME interface, depending upon bus band widthconsiderations. Host computer boards which may be employed in thepresent invention include, for example, Force SPARC/CPU-2E and HP9000Model 7471. The user interface can be, for example, a Unix/X-Windowenvironment. The image processing board can be, for example, based uponTexas Instruments' MVP and other chips to provide real-time imageaveraging, registration and other processing necessary to produce highquality difference images for intraoperative viewing. This board willalso drive a 120×1024 RGB display to show a sequence of differenceimages over time with pseudo-color mapping to highlight tumor tissue.Preferably, a second monitor is used for the host computer to increasethe overall screen real estate and smooth the user interface. Theprocessing board (fully programmable) can support a VME64 masterinterface to control data transactions with the other boards. Lastly, aperipheral control board can provide electrical interfaces to controlmechanical interfaces from the host computer. Such mechanical interfacescan include, for example, the light source and optical detector controlbox.

A real-time data acquisition and display system, for example, maycomprise four boards for acquisition, image processing, peripheralcontrol and host computer. A minimal configuration with reducedprocessing capabilities may comprise just the acquisition and hostcomputer boards. The acquisition board comprises circuitry to performreal-time averaging of incoming video frames and allow readout ofaveraged frames at a maximum rate bus. A VME bus is preferred because ofits high peak bandwidth and compatibility with a multitude of existingVME products. The acquisition board should also support many differenttypes of optical detectors via a variable scan interface. A daughterboard may support the interfacing needs of many different types ofoptical detectors and supply variable scan signals to the acquisitionmotherboard. Preferably, the unit comprises a daughter board interfacingto an RS-170A video signal to support a wide base of cameras. Othercamera types, such as slow scan cameras with a higher spatial/contrastresolution and/or better signal to noise ratio, can be developed andincorporated in the inventive device, as well as improved daughterboards to accommodate such improved cameras.

According to a preferred embodiment, data, such as analog video signals,are continuously processed using, for example, an image analyzer (e.g.,Series 151 Image Processor, Imaging Technologies, Inc. Woburn, Mass.).An image analyzer can receive and digitize an analog video signal withan analog to digital interface and perform such a function at a framespeed of about {fraction (1/30)}th of a second (e.g., 30 Hz or “videospeed”). Processing the signal involves first digitizing the signal intoa series of pixels or small squares assigned a value (in a binarysystem) dependent upon the number of photons (i.e., quantity of emr)being reflected off tissue from the part of the area of interestassigned to that pixel. For example, in a standard 512×512 image from aCCD camera, there would be 262,144 pixels per image. In an 8 bit system,each pixel is represented by 8 bits corresponding to one of 256 levelsof gray.

The signal processing means preferably includes a programmable look-uptable (e.g., CM150-LUT16, Imaging Technology, Woburn, Mass.) initializedwith values for converting gray coded pixel values, representative of ablack and white image, to color coded values based upon the intensity ofeach gray coded value. This can provide image enhancement via an imagestretch. An image stretch is a technique whereby the highest and lowestpixel intensity values used to represent each of the pixels in a digitalimage frame are determined over a region of the image frame which is tobe stretched. Stretching a selected region over a larger range of valuespermits, for example, easier identification and removal of relativelyhigh, spurious values due to noise (e.g., glare).

The processing means may further include a plurality of frame buffershaving frame storage areas for storing frames of digitized image datareceived from the A/D interface. The frame storage area comprises atleast one megabyte of memory space, and preferably at least 8 megabytesof storage space. An additional 16-bit frame storage area is preferredas an accumulator for storing processed image frames having pixelintensities represented by more than 8 bits. The processing meanspreferably includes at least three frame buffers, one for storing theaveraged control image, another for storing the subsequent image, and athird for storing a comparison image.

According to preferred embodiments, the processing means furthercomprises an arithmetic logic unit (e.g, ALU-150 Pipeline Processor) forperforming arithmetical and logical functions on data located in one ormore frame buffers. An ALU may, for example, provide image (data)averaging in real time. A newly acquired digitized image may be sentdirectly to the ALU and combined with control image stored in a framebuffer. A 16 bit result can be processed through an ALU, which willdivide this result by a constant (i.e., the total number of images). Theoutput from the ALU may be stored in a frame buffer, further processed,or used as an input and combined with another image.

Normally, areas of increased neuronal activity exhibit an increase ofthe emr absorption capacity of neuronal tissue (i.e., the tissue getsdarker if visible light is used for emr illumination, or an intrinsicsignal increases in a positive direction). Similarly, a decrease inneuronal activity is indicated a decrease of emr absorption capacity ofthe tissue (i.e., the tissue appears brighter, or intrinsic signalsbecome negative). For example, image A is a subsequent averaged imageand image B is an averaged control image. Normally, when a pixel inimage A is subtracted from a pixel in image B and a negative valueresults, this value is treated as zero. Hence, difference images cannotaccount for areas of inhibition. The present invention provides a methodfor identifying both negative and positive intrinsic signals, by: (a)subtracting image A (a subsequent averaged image) from image B (anaveraged control image) to create a first difference image, whereby allnegative pixel values are zero; and (b) subtracting image B from image Ato create a second difference image whereby all negative pixel valuesare zero; and adding the first and second difference images to create a“sum difference image.” The sum difference image shows areas ofincreased activity (i.e., color coded with warmer colors such as yellow,orange, red) and show areas of less activity or inhibition (i.e., colorcoded with colder colors such as green, blue, purple). Alternatively,one can overlay the first difference image on the second differenceimage. Either method provides an image of increased neuronal activityand decreased neuronal activity. The difference output may besuperimposed upon the real time analog video image to provide a videoimage of the area of interest (e.g., cortical surface) superimposed witha color-coded difference frame, in frozen time, to indicate where thereare intrinsic signals in response to some stimulus or paradigm.

The comparison (e.g., difference) data is, preferably, further processedto smooth out the image and remove high frequency noise. For example, alowpass spatial filter can block high spatial frequencies and/or lowspatial frequencies to remove high frequency noises at either end of thedynamic range. This provides a smoothed-out processed difference image(in digital format). The digitally processed difference image can becolor-coded by assigning a spectrum of colors to differing shades ofgray. This image is then converted back to an analog image (by an ADIboard) and displayed for a real time visualization of differencesbetween an averaged control image and subsequent images. Moreover, theprocessed difference image can be superimposed over the analog image todisplay specific tissue sites where a contrast enhancing agent may havea faster uptake, or where an intrinsic signal may be occurring.

Processing speed may be enhanced by adding a real time modular processoror faster CPU chip to the image processor. One example of a real timemodular processor which may be employed in the present invention is a150 RTMP-150 Real Time Modular Processor (Imaging Technology, Woburn,Mass.).

The processing means may further include an optical disk for storingdigital data, a printer for providing a hard copy of the digital and/oranalog data and a display, such as a video monitor to permit thephysician to continuously monitor the comparison data output.

A single chassis may house all of the modules necessary to provideoptical imaging according to the present invention. The necessarycomponents, whether or to whatever degree integrated, may be installedon a rack that is easily transportable within and between operating andhospital rooms along with display monitors and peripheral input andoutput devices.

Imaging Methods

Methods for imaging neuronal activity involve comparison of control datato data acquired during neuronal activity, inhibition or dysfunction.Neuronal tissue may be stimulated or inhibited without applying anyexternal influence. Seizures, strokes, neuronal dysfunction and tissuenon-viability are exemplary of such occurrences. Alternatively,intrinsic signals may be evoked by stimulating neuronal tissue usingdirect stimulation techniques or specific paradigms. Suitable paradigmsare well known in the art and include, for example, presenting picturesof objects to a patient and asking the patient to name the object. Suchnaming exercises alter neuronal activity and produce an associatedintrinsic signal.

An optical detector, such as a video CCD, is focused upon the area ofinterest during high intensity emr illumination. A first averaged imagemay be acquired, digitized and stored in a frame buffer. During animaging study, it is important to update the averaged image framefrequently to account for patient movement and for tissue movements dueto surgical manipulation. The area of interest is subsequently monitoredat regular intervals, or an appropriate paradigm is administered.Subsequent image frames are acquired and stored, and subtractivelycompared to produce difference images (preferably, one or two persecond) using the above-described processing means. The areas in whichneuronal activity has occurred are indicated in the difference image.The difference image can be stored to allow the surgeon to study thearea of interest in real time during an operation.

The present invention further provides a method for imaging of corticalfunctional areas and dysfunctional areas, such as those areas of severeepileptic activity. The method involves administering a paradigm toevoke an intrinsic signal for mapping a particular cortical function, oridentifying an area of hyperactivity that is the location of epilepticactivity in an epileptic patient. An epileptogenic area of the cortex isvisualized as spontaneously more active and can be imaged by theinventive apparatus by mapping intrinsic signals of cortical activity.Retinal function and dysfunction may also be detected and monitoredusing the optical imaging techniques described herein.

The inventive apparatus and method may also be employed to imageperipheral nerve damage and scarring. Nerves of the central andperipheral nervous system (PNS) are characterized by the ability toregenerate after damage. During operations to repair damaged peripheralor cranial nerves, one can image areas of nerve damage by imaging areasof blockage of intrinsic signals. For example, the nerve is exposed inthe area of interest and then stimulated upstream of the site of damage.The active nerve pathway is imaged by intrinsic signals in the processeddifference image after stimulation. The site of nerve damage or blockageis evidenced by an abrupt end or diminution to the intrinsic signal. Inthis way, the surgeon is able to obtain real time information on theprecise location of nerve damage and to correct the damage, if possible.

The imaging method may acquire data at the surface of an area ofinterest. As described above, longer wavelengths of emr (in the infraredrange) can be used to image areas of interest which are deeper in tissueor below overlying tissue. In some areas of the body longer wavelengthvisible light and near infrared emr can easily pass through such tissuefor imaging. Moreover, if a difference image is created between theimage acquired at 500 nm emr and the image acquired at 700 nm emr, thedifference image will show an optical slice of tissue. Administration ofan imaging agent which absorbs specific wavelengths of emr can act as atissue filter of emr to provide a filter in the area of interest. Inthis instance, it is desirable to utilize an imaging agent that remainsin the tissue for a prolonged period of time.

EXAMPLE 1

This example illustrates optical changes indicative of neuronal activityin a human subject by direct cortical electrical stimulation. Surfaceelectrical recordings (surface EEG, ECOG) were correlated with opticalchanges. Intrinsic optical changes were evoked in an awake patientduring stimulating-electrode “calibration”. Four stimulation trials weresequentially applied to the cortical surface, each stimulation evokingan epileptiform afterdischarge episode. A stimulation trial consistedof: 1) monitoring resting cortical activity by observing the output ofthe recording electrodes for a brief period of time, 2) applying anelectric current via the stimulation-electrodes to the cortical surfaceat a particular current for several seconds, and 3) monitoring theoutput of the recording electrodes for a period of time afterstimulation has ceased.

The cortex was evenly illuminated by a fiber optic emr passing through abeam splitter, controlled by a D.C. regulated power supply (Lambda,Inc.) and passed through a 695 nm longpass filter. Images were acquiredwith a CCD camera (COHU 6500) fitted to the operating microscope with aspecially modified cineadaptor. The cortex was stabilized with a glassfootplate. Images were acquired at 30 Hz and digitized at 8 bits(512×480 pixels, using an Imaging Technology Inc. Series 151 system,Woburn, Mass.). Geometric transformations were applied to images tocompensate for small amounts of patient motion (Wohlberg, DigitalImaging Warping, I.E.E.E. Computer Society, Los Alamitos, Calif., 1988).Subtraction of images collected during the stimulated state (e.g.,during cortical surface stimulation, tongue movement or naming) fromthose collected during a control state with subsequent division by acontrol image resulted in percentage difference maps. Raw data (i.e., nodigital enhancement) were used for determining the average opticalchange in specified regions (average sized boxes was 30×30 pixels or150-250 μm²). For pseudocolor images, a linear low pass filter removedhigh frequency noise and linear histogram transformations were applied.Noise was defined as the standard deviation of fluctuations insequentially acquired control images as 0.003-0.009.

A series of images (each image consisting of an average of 128 framesacquired at 30 Hz) were acquired during each of the four stimulationtrials. A current of 6 mA was used for the first three stimulationtrials, and 8 mA for the fourth. After a sequence of 3-6 averagedcontrol images were acquired, a bipolar cortical stimulation current wasapplied (either 6 mA or 8 mA) until epileptiform after dischargeactivity was evoked (as recorded by the surface electrode). Images werecontinuously acquired throughout each of the four stimulation trials.

The percentage change in absorption of light for each pixel wascalculated for each image acquired during the four stimulation trials.The average percentage changes over the four areas (indicated by thefour square regions marked in FIG. 1A) were plotted graphically in FIGS.1B, 1C, and 1D for comparison and analysis of the dynamic changesoccurring in these four spatial areas.

The optical changes between the stimulating electrodes (site #1, FIG.1A) and near the recording electrode (site #3) showed a graded responseto the intensity and duration of each afterdischarge episode (FIG. 1B).The spatial extent of the epileptiform activity was demonstrated bycomparing a baseline image collected before stimulation to thoseobtained immediately after stimulation. The intensity and spread of theoptical changes were much less following stimulation #2 (shortest leastintense afterdischarge episode) than after stimulation #4 (longest mostintense afterdischarge episode).

When the optical changes were below baseline, the surface EEG recordingsdid not identify epileptiform activity (n=3 patients). At site #3 inFIG. 2A1, the optical changes after stimulation were below baseline(i.e., black regions in FIG. 2A3). However, during the fourthstimulation, the epileptiform activity spread into the area of site #3and the optical signal did not go below baseline until later (site #3,FIG. 1B). This negative optical signal likely represents inhibitedneuronal populations (an epileptic inhibitory surround), decreasedoxygen delivery, or blood volume shunted to activated regions. The dataillustrated in FIGS. 1-4 also corresponds to Example 1.

EXAMPLE 2

Stimulation mapping of the cortical surface was performed on awake humanpatients under local anesthesia to identify sensory/motor cortex andBroca's areas. The illumination source and optical detection device andprocessing techniques used were the same as those described inExample 1. During three “tongue wiggling” trials, images were averaged(32 frames, 1 sec) and stored every 2 seconds. A tongue wiggling trialconsisted of acquiring 5-6 images during rest, then acquiring imagesduring the 40 seconds that the patient was required to wiggle his tongueagainst the roof of his mouth, and then to continue acquiring imagesduring a recovery period. The same patient was then required to engagein a “language naming” trial. A language naming trial consisted ofacquiring 5-8 images during rest (control images—the patient silentlyviewing a series of blank slides), then acquiring images during theperiod of time that the patient engaged in the naming paradigm (naming aseries of objects presented with a slide projector every 2 seconds,selected to evoke a large response in Broca's area), and finally aseries of images during the recovery period following the time when thepatient ceased his naming task (again viewing blank slides whileremaining silent). The results are shown in FIGS. 6 and 7 and describedin the description of those Figures

These results agree with those data reported by Lee et al. (Ann. Neurol.20:32, 1986), who reported large electrical potentials in the sensorycortex during finger movement. The magnitude of the optical changes inthe sensory cortex during tongue movement (10-30%) parallelssensory/motor cortex studies where cerebral blood flow increases 10-30%during motor tasks (Colebatch et al., J. Neurophysiol. 65:1392, 1991).Further, utilizing Magnetic Resonance Imaging (MRI) of blood volumechanges in human visual cortex during visual stimulation, investigatorshave demonstrated increases of up to 30% in cerebral blood volume(Belliveau et al., Science 254:716, 1991).

Optical images were obtained from this same cortical region (i.e., areaof interest) while the patient viewed blank slides and while namingobjects on slides presented every two seconds. Percentage differencemaps obtained during naming showed activation of the premotor area. Thesites of speech arrest and palate tingling were identified by surfacestimulation and demonstrate optical signals going in the oppositedirection. The area of activation was clearly different from that evokedby tongue movement without speech production. The optical images ofpremotor cortex activation during naming were in similar locations tothe cortical areas identified in PET single word processing studies(Peterson, et al., Nature 331:585, 1991; and Frith et al., J.Neuropsychologia 29:1137, 1991). The optical changes were greatest inthe area of the cortex traditionally defined as Broca's area and not inareas where electrical stimulation caused speech arrest.

EXAMPLE 3

Human cortex was imaged using the illumination source and opticaldetector described in Example 1. Functional mapping was conducted priorto and during imaging, as described with reference to FIGS. 8 and 9. Thedata illustrated in FIG. 8 demonstrates how a surgeon might use thisinvention intraoperatively to map language cortex and to avoidsurgically removing tissue having important functional properties. Thedata illustrated in FIG. 9 demonstrate that optical imaging can alsoidentify both essential and secondary language areas that must bepreserved during neurosurgical procedures.

EXAMPLE 4

Activation of sensory cortex by stimulation of a peripheral nerve wasimaged using a rat model. The results are shown in and described withreference to FIG. 5. This data demonstrates that the method andapparatus of the present invention may be used to map functional areasof the cortex providing afferent input while the subject isanesthetized.

EXAMPLE 5

Areas of interest can be imaged through intact tissues, such as bone,dura, muscle, connective tissue and the like. FIG. 10 illustratesidentification of a brain tumor through the intact cranium using opticalimaging techniques of the present invention. Tumor cells were injectedinto the left side of the intact cranium of a rat to cause developmentof a glioma on the left brain hemisphere. The right hemisphere wasnormal. FIGS. 10A-10D illustrate the dynamics and visibility of dyeperfusion in both the tumor and neuronal tissue. Neuronal activity maylikewise be imaged “through” intact tissues.

EXAMPLE 6

Optical contrast enhancing agents may be used in connection with opticalimaging techniques of the present invention. The utility of such agentsmay be demonstrated using hippocampal brain slice preparations.Hippocampal slices may be loaded in a chamber provided with artificialcerebral spinal fluid (“ACSF”), albumin labelled with indocyanine green(“ICG”) (approx. 2 mM) and 2% DMSO. After one hour, the tissue will bevisibly stained. Because the albumin-labeled ICG collects in theextracellular space, this staining technique may be used to detectchanges in neuronal activity and/or functions that are correlated tochanges in the volumes of the extracellular space. Similarly, thefluorescent agent Biodipy (available from Molecular Probes, Inc., P.O.Box 22010, Eugene, Oreg. 97402) bound to albumin will collect inextracellular space and may be used as a contrast enhancing agent todetect neuronal states or changes correlated to changes in the volume ofthe extracellular space.

We claim:
 1. A physiologically non-invasive method for detecting opticalchanges indicative of neuronal activity or dysfunction in an area ofinterest in a human patient, comprising: (a) illuminating the area ofinterest with an illumination source emitting electromagnetic radiation(emr) having at least one wavelength of from about 450 nm to about 2500nm; (b) detecting one or more optical properties of the area of interestwhen it is illuminated with the emr using a photon detector andacquiring and storing a control data set representing the one or moreoptical properties detected; (c) detecting one or more properties of thearea of interest at a time different from acquisition of the controldata set and acquiring a subsequent data set representing the one ormore optical properties detected during the different time; and (d)comparing the subsequent data set with the control data set to produce acomparison data set that identifies changes in the one or more opticalproperties and thereby identifies areas of neuronal activity ordysfunction.
 2. A method according to claim 1, additionally comprisinginducing neuronal activity in the area of interest by administering anelectrical stimulus to neuronal tissue.
 3. A method according to claim1, additionally comprising inducing neuronal activity in the area ofinterest by administering a paradigm that stimulates intrinsic neuronalactivity.
 4. A method according to claim 1, wherein neuronal activity isinduced by a natural occurrence.
 5. A method according to claim 1,wherein the illumination source and the photon detector are fixed inposition relative to one another.
 6. A method according to claim 1,wherein the illumination source and the photon detector are fixedrelative to one another and are mounted to the human patient.
 7. Amethod according to claim 1, wherein the area of interest includes aperipheral nerve.
 8. A method according to claim 1, wherein the area ofinterest comprises neuronal cortex.
 9. A method according to claim 1,wherein the photon detector is a video camera.
 10. A method according toclaim 1, additionally comprising amplifying portions of the control dataset to enhance a contrast of the comparison data set.
 11. A methodaccording to claim 1, additionally comprising identifying data points inthe comparison data set having intermediate values that representoptical changes indicative of neuronal activity, and mapping the datapoints having intermediate values to one of logarithmically and linearlyincreasing values to enhance the contrast of the comparison data set.12. A method according to claim 1, wherein the control data set is acontrol image, the subsequent data set is a subsequent image, and thecomparison data set is a comparison image.
 13. A method according toclaim 1, additionally comprising aligning corresponding spatiallocations in the control and subsequent data sets to produce thecomparison data set.
 14. A method according to claim 1, additionallycomprising mapping different pixel values comprising a comparison imageto color values to enhance a contrast of the comparison image.
 15. Amethod according to claim 14, wherein positive-going optical changes aremapped to a first color value and negative-going optical signals aremapped to a second color value.
 16. A method according to claim 1,comprising detecting one or more optical properties selected from thegroup consisting of: reflection; refraction; diffraction; absorption;scattering; birefringence; refractive index; and Kerr effect.
 17. Amethod according to claim 1, comprising continuously illuminating thearea of interest.
 18. A method according to claim 1, comprisingilluminating the area of interest non-continuously.
 19. A methodaccording to claim 18, comprising illuminating the area of interestusing a short pulse (time domain), pulsed time, or amplitude modulated(frequency domain) illumination source.
 20. A method according to claim1, comprising illuminating the area of interest uniformly.
 21. A methodaccording to claim 1, comprising illuminating the area of interest withan illumination source emitting emr having at least one wavelength inthe infrared range.
 22. A method according to claim 1, performedintraoperatively.
 23. A method for optically imaging changes indicativeof changes in neuronal activity in an area of interest in a humanpatient, comprising: (a) illuminating an area of interest with anillumination source emitting electromagnetic radiation (emr) having awavelength of from about 450 nm to about 2500 nm by connecting theillumination source to an illumination probe and implementing theillumination probe in the area of interest; (b) detecting one or moreoptical properties of the area of interest when it is illuminated withthe emr using a photon detector connected to a detection probe implantedin the area of interest that produces detection signals; (c) conveyingcontrol detection signals to a data processor and producing a controldata set representing the one or more optical properties detected; (d)detecting one or more optical properties of the area of interest duringneuronal activity, producing subsequent detection signals, conveying thesubsequent detection signals to the data processor, and producing asubsequent data set representing the one or more optical propertiesdetected during neuronal activity; and (e) comparing the subsequent dataset with the control data set to produce a comparison data set thatidentifies changes in one or more optical properties and therebyidentifies areas of neuronal activity.
 24. A method for identifyingspatial locations corresponding to functional properties of neuronaltissue in a human patient comprising: (a) illuminating neuronal tissuewith an illumination source emitting electromagnetic radiation (emr)having one or more wavelengths of from about 450 nm to about 2500 nm;(b) detecting one or more optical properties of identifiable spatiallocations of the neuronal tissue when it is illuminated and acquiring acontrol image that maps the one or more detected optical properties toidentifiable spatial locations of the neuronal tissue; (c) detecting oneor more optical properties of neuronal tissue during neuronalstimulation and acquiring a subsequent image that maps the one or moredetected optical properties during neuronal stimulation to identifiablespatial locations of the neuronal tissue; and (d) comparing the controlimage to the subsequent image to produce a comparison image thatspatially locates changes in neuronal activity.
 25. A method accordingto claim 24, additionally comprising inducing neuronal activity in theneuronal tissue by administering a paradigm that stimulates intrinsicneuronal signals.
 26. A physiologically non-invasive method foroptically imaging changes indicative of changes in retinal function in aretina in a human patient, comprising: (a) illuminating the retina withan illumination source emitting electromagnetic radiation (emr) havingat least one wavelength of from about 450 nm to about 2500 nm; (b)detecting one or more optical properties of the retina when it isilluminated with the emr using a photon detector and acquiring andstoring a control data set representing the one or more opticalproperties detected; (c) detecting one or more optical properties of theretina during retinal activity and acquiring a subsequent data setrepresenting the one or more optical properties detected during retinalactivity; and (d) comparing the subsequent data set with the controldata set to produce a comparison data set that identifies changes in theone or more optical properties and thereby identifies areas of retinaldysfunction.
 27. A method for monitoring the neuronal activity anddysfunction in a human patient, comprising: (a) illuminating neuronaltissue with an illumination source emitting electromagnetic radiation(emr) having at least one wavelength of from about 450 nm to about 2500nm; (b) detecting one or more optical properties of the illuminatedneuronal tissue and acquiring a baseline data set representing one ormore of the optical properties detected; (c) detecting one or moreoptical properties of the illuminated neuronal tissue at a timesubsequent to acquisition of the baseline data set and acquiring asubsequent data set; and (d) comparing the subsequent data set with thebaseline data set to identify changes in optical properties of theilluminated neuronal tissue that are indicative of neuronal activity ordysfunction.